This invention relates generally to human papilloma virus, and more particularly, relates to nucleotide sequences of short strands of human papilloma virus which can be amplified and/or used to determine the presence of human papilloma virus products in a test sample, and which also can be amplified and/or used to determine the specific type of human papilloma virus of types 6, 11, 16, 18, 31, 33 and 61 present in the test sample.
Human papilloma virus (HPV) is recognized as a venereally-transmitted disease of the anogenital tract which often is associated with the pathogenesis of cervical cancer and its precursor lesions. More than 56 types of HPV have been characterized. Of these, at least 21 types infect the anogenital tract. L. Gregoire et al., J. Clin. Micro 27 (12):2660-2665(1989). These mucosotropic viruses are associated most frequently with benign condyloma or latent infections. However, the presence of HPV in premalignant lesions and invasive cancers, particularly of the cervix, may reflect the oncogenic potential of these viruses. See P. M. Howley, in Important Advances in Oncology, D. T. DeVita, Jr. et al., eds., J. B. Lippincott, Philadelphia, Pa. (1987) at pages 55-73.
Certain HPV types, namely, HPV type 16 and type 18, and to a lesser extent HPV types 31, 33 and 35, are found in a high proportion of invasive cervical cancers and their metastases. However, many HPV types which infect the anogenital tract, such as HPV types 6 and 11, are found most commonly in benign condyloma and only rarely are found in invasive cancers. HPV detected in the anogenital tract can be classified broadly as low risk papilloma viruses (HPV types 6 and 11), intermediate risk papilloma viruses (HPV types 31, 33 and 35) or high risk papilloma viruses (HPV types 16 and 18), based on the association of the particular HPV type with malignancy. A. T. Lorincz et al., J. Nat'l. Cancer Inst. 79:671 (1987). Thus, the detection of the presence of HPV and the determination of the specific type of HPV can provide a diagnostic and prognostic tool useful for determining the clinical significance associated with certain HPV types. The early detection of HPV by sensitive and specific reagents and methodologies also could provide earlier therapeutic management and counseling.
A need therefore exists for accurate and reliable methods to identify and type HPV in clinical specimens. However, known polyclonal antisera prepared by immunizing animals with disrupted virions are capable of detecting HPV antigens in only about 30-70% of cutaneous and mucosal warts. Further, the antisera are broadly cross-reactive. Available immunological tests have two major drawbacks. First, only well-differentiated cells apparantly are capable of viral antigen expression. HPV-infected tissues which show higher degrees of neoplasia, such as carcinoma in situ, rarely contain HPV antigen. Thus, the further the development of the malignancy, the smaller the amount of detectable virus in the tested tissue. Secondly, these immunological tests are unable to identify specific viral types.
It is known that papilloma viruses share amino acid sequences in the major capsid proteins. See, for example, C. C. Baker, in The Papovaviridae (Vol. 2), P. M. Howley and N. P. Salzman, eds., Plenum Publ. Corp., New York (1987) at pages 321-385. The DNAs of this virus cross-hybridize, indicating homologous sequences. M. F. Law et al., J. Virol. 58:225-229 (1979). Thus, molecular hybridization techniques have been developed as a more sensitive and specific means of detecting and differentiating HPV DNA and RNA in clinical specimens. See A. T, Lorinez, Obstetrics and Gynecol, Clinics of N. America 14:451 (1987).
Sequences specific for the DNA and RNA of human papilloma virus are known and have been published. See, for example, PCT application No. WO 89/69940 published Oct. 19, 1989, PCT application No. WO 86/05816 published Oct. 9, 1986 and European Patent Application No. 0 301 968 published Feb. 1, 1989.
The molecular hybridization techniques used to detect homologous DNA sequences are sensitive and can be highly specific if used with probes which bind to nucleic acid sequences which are unique to a particular HPV type. However, the concentration of total viral DNA in a given clinical sample may be below the limit of sensitivity of the test. For example, the amount of viral DNA in dysplastic cervical lesions is reduced with increasing dysplasia.
To overcome this problem of sensitivity, viral DNA sequences can be amplified by using, for example, the polymerase chain reaction (PCR) or the ligase chain reaction (LCR) techniques. The products thus obtained can be identified by using conventional hybridization techniques for identification of virus types, such as Southern blotting. See C. Oste, Biotechniques 6:163 (1988), K. B. Mullis, U.S. Pat. No. 4,683,202, and EP-A-320 308 (BioTechnica).
Both PCR and LCR serve to amplify the DNA present in a test sample to detectable levels. In practice, the level of sensitivity is about 50 to 100 copies per sample. The next most sensitive technique is dot-blot, which can detect about 10,000 molecules, while Southern blot reliably detects about 100,000 copies of DNA per sample.
Thus, the appropriate diagnosis of HPV may require two steps. In one strategy, the presence of a clinically relevant type of HPV is first detected with a group-specific primer. After the presence of HPV is detected, differentiation between types can be performed by using a type-specific probe having low homology between the HPVs of the group. Alternatively, differentiation can be performed using a mixture of type-specific probes at the outset, provided these probes amplify DNA independently of each other, and that they can be detected independently. In the past, such tasks were attempted using specific antibodies. In general, nucleic acid probes and primers allow greater discrimination among subtypes than do antibodies. The use of DNA-based tests increases both sensitivity and specificity over prior-art antibody-based tests.
It therefore would be advantageous to provide oligonucleotide strands of DNA which could be amplified and used to detect the presence, if any, of HPV in a test sample. It also would be advantageous to provide short oligonucleotide strands of DNA which could be amplified and used to detect the presence, if any, of specific types of HPV in the test sample. The combined use of oligonucleotide strands would be advantgeous for allowing for the specific and sensitive in vitro diagnosis of the presence and specific type of HPV present in test samples.